Golgi: Methods and Protocols

Preface
Contents
Contributors
Part I: Model Systems Used to Study Golgi Organization and Function
Chapter 1: Live Cell Imaging of Yeast Golgi Dynamics
1 Introduction
2 Materials
2.1 Equipment
2.2 Supplies
2.3 Media
3 Methods
3.1 Yeast Growth and Culture Preparation
3.2 Imaging
3.3 Data Analysis
4 Notes
References
Chapter 2: Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole
1 Introduction
2 Materials
2.1 Yeast and Vacuolar Stains
2.2 Microscopy
2.3 Software
3 Methods
3.1 Vacuolar Membrane Staining
3.2 Vacuolar Lumen Staining
3.3 Slide Preparation and Image Acquisition
3.4 Preparing Final Images for Analysis
3.5 Cell Selection from Acquired Images
3.5.1 Selecting Grouped Cells and Creating Single Plane Images
3.5.2 Regions of Interest (ROIs)
3.6 Isolating Signal over Background Through Thresholding
3.6.1 Multi-Otsu Thresholding
3.6.2 Auto Thresholding
3.7 Quantification of Colocalization Through Manders´ Coefficient
4 Notes
References
Chapter 3: Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster
1 Introduction
2 Materials
2.1 Dissection
2.2 Fixation
2.3 Permeabilization and Antibody Staining
2.4 Mounting
2.5 Imaging
3 Methods
3.1 Dissection and Fixation
3.2 Permeabilization and Antibody Staining
3.3 Mounting
3.4 Confocal Imaging Using Point Scanning Nikon A1R Confocal Microscope
3.5 Imaging Using Super-resolution Zeiss Elyra PS1 Structured Illumination Microscope
4 Notes
References
Chapter 4: Microscopy and Immunocytochemistry-Based Methods to Study Cell Wall Biosynthetic Enzymes in the Golgi
1 Introduction
2 Materials
2.1 Enzyme Localization by Confocal Microscopy
2.1.1 Cultivation of Nicotiana tabacum
2.1.2 Agrobacterium Transformation
2.1.3 Transient Expression in N. tabacum
2.1.4 Confocal Microscopy
2.1.5 BFA Treatment
2.2 Cell Wall Dot Blotting
2.2.1 Preparation of Alcohol Insoluble Residue (AIR)
2.2.2 Removing Starch from AIR
2.2.3 Extracting Cell Wall Using 1-M or 4-M KOH
2.2.4 Generating a Series of Twofold Dilutions and Sample Spotting
2.2.5 Dot Blotting Using Antibody
3 Methods
3.1 Enzyme Localization by Confocal Microscopy
3.1.1 Growing N. tabacum for Transient Expression
3.1.2 Transformation into Agrobacterium (GV3101)
3.1.3 Transient Expression in N. tabacum Leaf
3.1.4 Confocal Microscopy
3.1.5 BFA Treatment
3.1.6 Verification of the Fusion Protein
3.2 Cell Wall Dot Blotting
3.2.1 Preparation of Alcohol Insoluble Residue (AIR)
3.2.2 Removing Starch from AIR
3.2.3 Extracting Cell Wall Using KOH
3.2.4 Generating Twofold Dilutions and Sample Spotting
3.2.5 Dot Blotting Using Antibodies for Cell Wall Components
4 Notes
References
Chapter 5: Reconstitution of Golgi Biogenesis in Permeabilized Trypanosoma brucei Cells
1 Introduction
2 Materials
2.1 Cell Culture Media and Supplements
2.2 Buffers and Solutions
2.3 T. brucei Cell Lines
2.4 Imaging
3 Methods
3.1 Maintenance of T. brucei Cells
3.2 Preparation of T. brucei Cytosol
3.3 Digitonin Permeabilization of Live T. brucei Cells
3.4 Photoactivation and Time-Lapse Imaging of Digitonin-Permeabilized Cells
3.5 Image Analysis of Live Cell Microscopy Data
4 Notes
References
Chapter 6: Generation of GM130 Conditional Knockout Mouse
1 Introduction
2 Materials
2.1 Generation and Expression Reagents
2.2 Cell Culture, Transfection, and Transduction Reagents
2.3 Reagents for Blastocyst Injection, Uterine Transfer, and Mouse Genotyping
2.4 Equipment, Tools, and Supplies
3 Methods
3.1 Preparation of Construction
3.1.1 Purification of BAC from E. Coli
3.1.2 Preparation of Competent Cells EL350
3.1.3 Electroporating BAC to EL350
3.1.4 Preparation of EL350 Electro-Competent Cells for Recombination
3.2 Construction of the Targeting Vector
3.2.1 Construction of PL253 Retrieving Vector
3.2.2 Retrieving
3.2.3 Insertion of the First loxP Site by PL452-GM130 Mini-Targeting Vector
3.2.4 Insertion of the Second loxP Site by PL451-GM130 Mini-Targeting Vector
3.3 Stem Cell Targeting
3.3.1 Preparation of Target Carrier before Electroporation
3.3.2 Preparation of MEF Cells
3.3.3 Preparation of Stem Cells before Electroporation
3.3.4 Electroporation of ES Cells
3.4 Blastocyst Injection of Stem Cells
3.4.1 Preparation of Stem Cells
3.4.2 Preparation of Blastocysts
3.4.3 Blastocyst Microinjection with Embryonic Stem Cells
3.4.4 Uterine Transfer
3.4.5 DNA Extraction for Identification of the Mouse Genotypes
3.4.6 Genotypes by PCR
3.5 Cultivation of Mice
4 Notes
References
Chapter 7: Monitoring Effects of Membrane Traffic Via Changes in Cell Polarity and Morphogenesis in Three-Dimensional Human Pl...
1 Introduction
2 Materials
2.1 Culturing Cells and Cysts
2.2 Fixing and Staining Cysts
3 Methods
3.1 Preparing a Healthy Monolayer
3.2 Preparing Chamber Dishes
3.3 Plating Single-Cell Suspension
3.4 Generating Cysts with Geltrex Overlays
3.5 Fixing with Paraformaldehyde (See Note 6)
3.6 Permeabilizing Cells with SDS (See Note 9)
3.7 Fixing hPSC-Cysts with Methanol
3.8 Permeabilizing Cells with Triton
3.9 Staining Cells
3.10 Imaging
4 Notes
References
Part II: Morphological Studies of Golgi Organization and Function
Chapter 8: Immunofluorescence Microscopy of the Mammalian Golgi Apparatus
1 Introduction
2 Materials
2.1 Culture Dishes, Coverslips, and Slides
2.2 Chemical Reagents
2.3 Tissue Culture Cells
3 Methods
4 Notes
References
Chapter 9: Studying the Organization of the Golgi by Super-Resolution Microscopy
1 Introduction
2 Materials
2.1 Antibodies and Plasmid Constructs
2.2 Cell Culture and Transfection
2.3 Immunofluorescence
2.4 Microscopy
2.5 Softwares
3 Methods
3.1 Sample Preparation and Imaging of Fixed Cells
3.1.1 Coverslips Sterilization
3.1.2 Seeding, Transfection, and Nocodazole Treatment of HeLa Cells
3.1.3 Immunofluorescence
3.2 Image Acquisition Under Airyscan Microscopy
3.3 Identification of En Face and Side Views of Ministacks
3.4 En Face Averaging
3.5 Calculation of the Radius of a Golgi Protein
3.6 Analysis of the Organization of Native Golgi Stacks
4 Notes
References
Chapter 10: Super-Resolution Live Imaging of Cargo Traffic Through the Golgi Apparatus in Mammalian Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Gene Transfection and RUSH Experiment
2.3 SCLIM-1 Setup
2.4 SCLIM-2K Setup
2.5 Image Analyses
3 Methods
3.1 Cell Culture and Transfection
3.2 SCLIM Observation
3.3 Image Reconstruction and Processing by Volocity
3.4 Image Processing by NIS-Elements
4 Notes
References
Chapter 11: Studying Golgi Structure and Function by Thin Section TEM
1 Introduction
2 Materials
2.1 TEM Sample Preparation
2.2 Imaging and Quantitation
2.3 DAB Cytochemical Staining
2.4 Pre-embedding Immuno-EM
3 Methods
3.1 TEM Sample Preparation: Flat Embedding, Trimming the Pyramid, Sectioning, and Post-staining
3.2 Acquiring TEM Micrographs with Systematic Random Sampling
3.3 Anonymization of TEM Micrographs Using the Microscopy Image Browser Software
3.4 Analyzing Golgi Morphology from Thin TEM Sections
3.5 DAB Cytochemical Staining
3.6 Pre-embedding Immunolabelling
4 Notes
References
Chapter 12: Algorithm for Modern Electron Microscopic Examination of the Golgi Complex
1 Introduction
2 Materials
2.1 Instruments
2.2 Working Solutions
2.3 Reagents and Software
3 Methods
3.1 Decision Tree: Selection of EM Technique for the Analysis of the GC
3.2 Serial Section Collection Protocol (Adapted from Ref.)
3.3 Perfusion Fixation
3.4 The Silver Filler Prevention of Charging
3.5 Pre-embedding and NPG-Silver Enhancement (Modified from Ref. [25])
3.6 Gold Enhancement with Gum Arabic (Adapted from Ref.)
3.7 Preparation of Samples Containing Both Control and Experimental Cells
4 Notes
References
Chapter 13: High-Pressure Freezing Followed by Freeze Substitution: An Optimal Electron Microscope Technique to Study Golgi Ap...
1 Introduction
2 Materials
2.1 High-Pressure Freezing
2.2 Freeze Substitution
2.3 Infiltration, Embedding, and Sectioning
2.4 Post-Staining and Imaging
3 Methods
3.1 Cell Monolayer Culture
3.2 High-Pressure Freezing
3.3 Freeze Substitution
3.4 Infiltration and Embedding
3.5 Sectioning and Imaging
3.6 Post-Sectioning Staining
3.7 Identify Golgi Apparatus under Electron Microscope
4 Notes
References
Chapter 14: Negative Staining Electron Microscopy for Morpho-Functional Characterization of Purified Golgi Membranes from Mamm...
1 Introduction
2 Materials
2.1 Golgi Purification Material
2.2 In Vitro Morpho-Functional Assay Materials
2.3 NS Materials
2.4 Image Acquisition
2.5 Software
3 Methods
3.1 Purification of Golgi Membranes from HeLa Cells
3.2 In Vitro Morpho-Functional Assay
3.3 NS Procedure
3.4 Morpho-Functional Analysis
4 Notes
References
Chapter 15: An Electron Tomographic Analysis of Giantin-Deficient Golgi Proposes a New Function of the Golgin Protein Family
1 Introduction
2 Materials
2.1 0.1 M Sodium Phosphate Buffer, pH 7.4 (PB)
2.2 Epon Resin
3 Methods
3.1 RNA Interference (RNAi) of Giantin
3.2 Sample Fixation for Electron Tomography
3.3 Sample Embedding for Electron Tomography
3.4 Sectioning and Data Collection
3.5 Construction of a Tomogram and 3D Model
3.6 Data Presentation with Quantitation
4 Notes
References
Chapter 16: Modeling the Cryo-EM Structure of the Exocyst Complex
1 Introduction
2 Materials
2.1 Software and Servers
2.2 Cryo-EM Map
2.3 Crystal Structures of Yeast Exocyst Subunits
2.4 Chemical Cross-Linking Mass Spectroscopy (CXMS) Data
3 Methods
3.1 Docking of Known Crystal Structures
3.2 3D Structure Prediction and Docking
3.2.1 3D Structure Prediction of Sec10, Sec15, and Exo84
3.2.2 Docking of Sec10, Exo84, and Sec15
3.3 de Novo Modeling
3.3.1 Secondary Structure Prediction
3.3.2 de Novo Modeling of Subunits with Docked Domains
3.3.3 de Novo Modeling of Sec5, Sec8, and Sec3
3.4 Model Validation
3.4.1 Validation with CXMS Data
3.4.2 Model Refinement and Validation
4 Notes
References
Chapter 17: Quantitatively Assessing Co-Localization of Golgi Proteins by Distance Analysis Using the DiAna Software
1 Introduction
2 Materials
2.1 Fluorescently Labeled Specimens
2.2 Microscopy
2.3 Software
2.3.1 ImageJ
2.3.2 Required Additional Components
2.3.3 DiAna
2.3.4 Spreadsheet and Other Statistical Software
3 Methods
3.1 Microscopy
3.2 DiAna Object-Based Colocalization Analysis
3.2.1 General Overview of DiAna Workflow
3.2.2 Object Recognition
3.2.3 Distance Analysis
3.2.4 Managing, Interpreting, and Presenting the Data
4 Notes
References
Chapter 18: A Primer on Deep Learning-Based Cellular Image Classification of Changes in the Spatial Distribution of the Golgi ...
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Alcian Blue-Coated Coverslips
3.2 Plating Cells on Alcian Blue-Coated Coverslips
3.3 Fixation with Cold Methanol and Rehydration
3.4 Blocking, Staining, Mounting, and Sealing
3.5 Image Acquisition
3.6 Deep Learning
3.7 Image Classification of Golgi
4 Notes
References
Part III: Biochemical and In Vitro Reconstitution Assays in Golgi Studies
Chapter 19: Reconstitution of Vesicle Budding from the TGN and Immunoisolation of Vesicles Enriched with a Specific Cargo Clie...
1 Introduction
2 Materials
2.1 PBS
2.2 1x KOAc Buffer
2.3 10x KOAc Buffer
2.4 Digitonin
2.5 10 mM GTP
2.6 ATP Regeneration System
2.7 Buffer E
2.8 PEI
2.9 Elution Buffer
2.10 SDS-PAGE Loading Buffer
2.11 SDS-PAGE Gel
2.12 SDS Running Buffer
2.13 Transfer Buffer
2.14 PBST
2.15 Rotors, Centrifuges, and Ultracentrifuge Tubes
2.16 Other Materials
3 Methods
3.1 Preparing the Cytosol from Rat Liver
3.2 Preparation of the Cytosol from Mammalian Cells
3.3 Preparing Digitonin-Permeabilized Cells (Fig. 1, Step A)
3.4 Reconstituting the Release of Cargo Proteins into Transport Vesicles at the TGN (Fig. 1, Step A)
3.5 Immunoisolating TGN-Derived Vesicles Enriched with a Specific Transmembrane Cargo (Fig. 1, Step B)
3.6 Immunoblotting
4 Notes
References
Chapter 20: Common Assays in Mammalian Golgi Studies
1 Introduction
2 Materials
2.1 Common Reagents
2.2 Tools Used in RNA-seq Bioinformatic Analysis
2.3 Tools for RNA-seq Analysis and Database Search
2.4 Visualization Tools for RNA-seq Analysis
3 Methods
3.1 Protein Expression, Depletion, and Turnover Assay
3.1.1 Protein Expression by Transient Transfection
3.1.2 Establishment of Stable Cell Lines Using Viral Systems
3.1.3 Gene Knockdown
3.1.4 Cycloheximide (CHX) Treatment to Determine Protein Turnover
3.2 Imaging Techniques for Morphological Studies
3.2.1 Immunofluorescence Microscopy
3.2.2 Electron Microscopy (EM)
3.2.3 EM Tomography
3.3 Common Assays to Study Golgi-Mediated Trafficking
3.3.1 Brefeldin A Treatments and Washout
3.3.2 Temperature Block and Release of VSV-G tsO45
3.3.3 Synchronized Release of Cargo Proteins Using the Retention Using Selective Hooks (RUSH) System
3.3.4 Pulse-Chase Labeling and Endo H Treatment
3.4 Subcellular Fractionation and In Vitro Reconstitution Using Isolated Golgi Membranes
3.4.1 Differential Centrifugation to Separate Different Cellular Structure
3.4.2 Equilibrium Gradients to Fractionate Membrane Organelles
3.4.3 Purification of Golgi Membranes from Animal Tissues
3.4.4 In Vitro Budding Assay
3.4.5 In Vitro Reconstitution of Golgi Disassembly and Reassembly During Cell Division
3.4.6 In Vitro Reconstitution of Golgi Structure Formation and Function in Semi-Permeabilized Cells
3.5 Systems Approaches in Golgi Studies
3.5.1 Proteomic and Secretomic Analysis
3.5.2 RNA-seq and Transcriptomic Analysis
3.5.3 Proximity Labeling Using BioID, TurboID, and Apex
4 Notes
References
Chapter 21: In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon During the G2-M Transition of the Cell Cycle
1 Introduction
2 Materials
2.1 General Reagents
3 Methods
3.1 Cell Permeabilization Assay to Investigate Mitotic Golgi Fragmentation
3.1.1 Preparation of Mitotic Cytosol
3.1.2 Preparation of Interphase Cytosol
3.1.3 Plasma Membrane Permeabilization of NRK Cells
3.1.4 Incubation of Permeabilized NRK Cells with Cytosol
3.2 Investigation of the Golgi Structure during G2/M Transition
3.2.1 Synchronization Protocols
3.2.2 Measure of the Various Cell Cycle Phases
3.3 Evaluation of Golgi Continuity during G2
3.4 Microinjection Assay in Intact Cells to Study Mitotic Golgi Fragmentation
4 Notes
References
Chapter 22: Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines
1 Introduction
2 Materials
2.1 Cells and Media (See Note 1)
2.2 Neon Transfection System (Thermo Fisher, Catalog No. MPK10096) to Transfect hTERT-RPE1 Cells
2.3 Lentivirus Production and Cell Transduction
2.4 Validation of VPS54-KO and VPS54-KO-R Cells
2.4.1 Western Blot
2.4.2 Immunofluorescence (IF) Microscopy
2.4.3 Cathepsin D Secretion Assay
3 Methods
3.1 Knocking out VPS54 in hTERT-RPE-1 Cells
3.2 Making hTERT RPE1 VPS54-KO-R Cells by Lentiviral Transduction
3.3 Validation of VPS54-KO and VPS54-KO-R Cells by WB
3.4 Validation of VPS54-KO and VPS54-KO-R Cells Rescue by IF
3.5 Validation of VPS54-KO and Rescue by Cathepsin D Secretion Assay
4 Notes
References
Chapter 23: Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Expression Plasmid
2.3 DNA Preparation and Recombination
2.4 Polymerase Chain Reaction (PCR)
2.5 Transfection and Transduction Reagents
2.6 Validation (Lysate Preparation, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, and Western Blot Components)
2.7 Validation (Immunofluorescence Experiment Reagents and Confocal Microscopy)
2.8 Antibodies
2.9 Materials for Degradation System
3 Methods
3.1 Generation of COG4 KO Cell Line Expressing OsTIR1
3.1.1 Plasmid Transformation and Preparation
3.1.2 Production of OsTIR1-9myc Retrovirus
3.1.3 Retroviral Transduction of OsTIR1-9myc into RPE1 COG4 KO Cells
3.1.4 Selection and Isolation of RPE1 COG4 KO Single Clones Expressing OsTIR1
3.1.5 Screening for OsTIR1-9myc Expressing Clones by WB Analysis
3.1.6 Screening for Single Clones That Uniformly Express OsTIR1 by IF Analysis
3.2 Generation of COG4 KO-OsTIR1 Expressing AID-Tagged COG4 Stable Cell Line
3.2.1 PCR of the mAID-mCherry
3.2.2 Cloning COG4-mAID-mCherry in pEntra1A
3.2.3 Creation of Expression Clone COG4-mAID-mCherry in pLenti
3.2.4 Production of COG4-mAID-mCherry Lentivirus
3.2.5 Lentiviral Transduction of COG4-mAID-mCherry into RPE1 COG4 KO TIR1-9myc Cells
3.2.6 Selection and Isolation of RPE1 COG4 KO Tir1-9myc Single Clone Expressing COG4-mAID-mCherry
3.2.7 Screening for COG4-mAID-mCherry Expressing Clones by WB Analysis
3.2.8 Screening for COG4-mAID-mCherry Expressing Single Clones by IF Analysis
3.2.9 Screening for the Clones Rescuing the COG4 KO Phenotypes
3.3 Testing Inducible Depletion of COG4-mAID Protein by Auxin (IAA)
3.3.1 WB Assay
3.3.2 IF Assay
4 Notes
References
Chapter 24: Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction
1 Introduction
1.1 Overview
1.2 Parts of the System-Plasmids and Packaging Cell Line
2 Materials
2.1 Cell Culture
2.2 Plasmids
2.3 Plasmid Amplification
2.4 Transfection and Transduction
2.5 Validation
2.6 Solutions
3 Methods
3.1 Safety When Handling Viral Vectors (See Note 1)
3.2 Plasmid Amplification and Isolation
3.3 Phoenix Cell Transfection
3.4 HeLa Cell Transduction
3.5 Check Transduction Efficiency
3.6 Puromycin Selection of Transduced HeLa Cells
3.7 Validation by Western Blot
3.8 Lentiviral Transduction of GRASP65-GFP
4 Notes
References
Chapter 25: Quantitative Proteomics Analysis of Purified Rat Liver Golgi
1 Introduction
2 Materials
2.1 Purification of Rat Liver Golgi Membranes
2.2 Golgi Protein Solubilization, Tryptic Digestion, and Peptide Labeling
2.3 Peptide Fractionation by OFFGEL Isoelectric Focusing
2.4 Offline LC-MALDI-MS/MS
2.5 Database Searching, Quantification, and Categorization of the Identified Proteins
2.6 Confirmation of the Proteomic Analysis by Western Blotting
3 Methods
3.1 Purification of Golgi Membranes from Rat Liver
3.2 Golgi Protein Solubilization, Tryptic Digestion, and Peptide Labeling
3.3 Peptide Fractionation by OFFGEL Isoelectric Focusing
3.4 Offline LC-MALDI-MS/MS
3.5 Database Searching, Quantification, and Categorizing the Identified Proteins
3.6 Confirmation of the Proteomic Results by Western Blotting
4 Notes
References
Chapter 26: Comparative Genomics for Evolutionary Cell Biology Using AMOEBAE: Understanding the Golgi and Beyond
1 Introduction
1.1 Evolution of MTS and Golgi Apparatus
1.2 Why AMOEBAE?
1.3 Applicability and Research Questions
1.4 Prerequisite Skills
2 Code and Data Availability
3 Method
3.1 Overall Approach
3.2 Installation
3.2.1 Computational Resources
3.2.2 Dependency Installation
3.2.3 Acquiring a Copy of the AMOEBAE Code
3.3 Workflow Configuration
3.3.1 Genome Sequence Files
3.3.2 Query Sequence Files
3.3.3 Sequence Similarity Search Parameters
3.3.4 Output Plot Organization
3.4 Running the Workflow
3.4.1 Selecting Reference Sequences
3.4.2 Executing Searches in All the Genomes
3.5 Interpreting Results
4 Limitations, Pitfalls, and Mitigation Strategies
5 Final Conclusions
References
Chapter 27: Common Markers and Small Molecule Inhibitors in Golgi Studies
1 Introduction
2 Overview of Chemical Tools for Manipulating the Golgi
2.1 Cell Cycle Modulators
2.2 Cytoskeleton Modulators
2.3 Protein Synthesis Inhibitors
2.4 Proteasomal Inhibitors
2.5 Lysosomal and Cytoplasmic Protease Inhibitors
2.6 Membrane Trafficking Modulators
2.7 Protein Glycosylation Inhibitors
2.8 Autophagy Inhibitors and Inducers
2.9 Golgi Stressors
2.10 ER Stressors and Ca2+ Modulators
2.11 Apoptosis Inducers and Inhibitors
2.12 ROS Inducers and Inhibitors
2.13 Secretase Inhibitors
2.14 Protein Kinase and Phosphatase Inhibitors, AAA ATPases Inhibitors
3 Materials
3.1 Cell Culture
3.2 Analysis by Immunofluorescence Microscopy
3.3 Analysis by Western Blot
3.4 Flow Cytometry
4 Methods
4.1 Cell Treatment, Sample Preparation, and Western Blot
4.1.1 Treatment of Cells with Small Molecules
4.1.2 Sample Preparation
4.1.3 SDS-PAGE and Western Blot
4.2 Immunofluorescence Microscopy
4.2.1 Standard IF Protocol
4.2.2 Coverslip Coating
4.3 Lectin Blot and Staining
4.3.1 Lectin Blot
4.3.2 Lectin Microscopy and Flow Cytometry
4.4 Glycosidase Digestion
5 Notes
References
Part IV: Mechanism of Golgi Structure Formation and Membrane Trafficking
Chapter 28: Visualizing Reversible Cisternal Stacking in Budding Yeast Pichia pastoris
1 Introduction
2 Materials
2.1 Microscopic Instruments and Software
2.1.1 Yeast Strains
2.1.2 Yeast Growth
2.1.3 Live-Cell Imaging
3 Methods
3.1 Creating the Yeast Strains
3.2 Preparing the Cells for Microscopy
3.3 Microscopic Acquisition
3.4 Image Processing
3.4.1 Filtering
3.4.2 Deconvolution
3.4.3 Making 4D Movie
3.5 4D Movie Analysis and Calculation of the TGN Peeling Frequency
4 Notes
References
Chapter 29: Methods for Studying Membrane-Proximal GAP Activity on Prenylated Rab GTPase Substrates
1 Introduction
2 Materials
2.1 Liposome Preparation
2.2 Rab-GAP Activity Assay
3 Methods
3.1 Liposome Preparation
3.1.1 Day 1
3.1.2 Day 2
3.2 Rab-GAP Activity Assay
3.2.1 Nucleotide Exchange
3.2.2 Rab-GAP Recruitment
3.2.3 Liposome Flotation and Recovery
3.2.4 Visualization of Reaction Outcome
4 Notes
References
Chapter 30: Studying the Role of Lipid Geometry in COPI Vesicle Formation
1 Introduction
2 Materials
2.1 Golgi Membrane Preparation
2.2 In Vitro COPI Vesicle Formation
2.3 Liposome Preparation
2.4 Electron Microscopy
2.5 In Vivo COPI Transport Assay
3 Methods
3.1 Golgi Membrane Preparation
3.2 Reconstituting COPI Vesicle Formation at an Arrested State
3.3 Rescuing the Arrest in COPI Vesicle Formation
3.4 Liposome Deformation Assay
3.5 Cell-Based COPI Transport Assay
4 Notes
References
Chapter 31: Unbiased Quantification of Golgi Scattering and Golgi-Centrosome Association
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunostaining
2.3 Confocal Microscope and Analysis
3 Methods
3.1 Cell Maintenance
3.2 Immunostaining
3.3 Image Acquisition/Parameters
3.4 Preparing Images for Analysis
3.5 Obtaining Golgi Coordinates Using ImageJ
3.6 Obtaining Centrosome Coordinates Using Imaris
3.7 Calculating Golgi-Golgi Distance and Golgi-Centrosome Distance Using MATLAB
4 Notes
References
Chapter 32: Detection and Analysis of Microtubule Nucleator γ-Tubulin Ring Complex
1 Introduction
2 Materials
2.1 Cell Culture and Treatments
2.2 Immunofluorescence Labeling and Microscopy
2.3 Isolation of Golgi Membranes
2.4 In Vitro Assay of γTuRC-Mediated Microtubule Nucleation
3 Methods
3.1 Detecting Golgi-Associated γTuRC Proteins
3.1.1 Immunostaining of γ-Tubulin and PolD1 on the Golgi
3.1.2 Immunoblotting of PolD1 on Golgi Membranes
3.2 Assaying Golgi-Associated Microtubule Nucleation
3.2.1 Regrowth of Golgi-Derived Microtubules After Cold-Induced Depolymerization
3.2.2 Regrowth of Golgi-Derived Microtubules After Nocodazole Washout
3.3 Assessing Microtubule-Nucleating Activity of Isolated γTuRCs
4 Notes
References
Chapter 33: Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes
1 Introduction
2 Materials
2.1 Dual-Color Live Cell Imaging of Synchronized Protein Trafficking with a Golgi Marker
2.2 Computer-Based Quantification of Golgi Traffic
3 Methods
3.1 Dual-Color Live Cell Imaging of Synchronized Protein Trafficking with a Golgi Marker
3.2 Quantifying Golgi Kinetics by Measuring Fluorescence Intensity Around Golgi Region
3.3 Quantification of Kinetics of Golgi Trafficking by Co-localization with Golgi Markers
4 Notes
References
Chapter 34: CARTS Formation Assay
1 Introduction
2 Materials
3 Methods
3.1 Establishment of HeLa Cells Stably Expressing mKate2-FM4-PAUF
3.1.1 Retrovirus Preparation
3.1.2 Retroviral Infection of HeLa Cells
3.2 CARTS Formation Assay
3.3 Quantification of CARTS Using ImageJ
4 Notes
References
Chapter 35: Quantification of Protein Exit at the Trans-Golgi Network
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmids
2.3 Equipment
2.4 Software
3 Methods
3.1 Cell Culture, Transfection, and Imaging Procedure
3.2 Imaging Analysis
4 Notes
References
Chapter 36: Endosomal Transport to Lysosomes and the Trans-Golgi Network in Neurons and Other Cells: Visualizing Maturational ...
1 Introduction
2 Materials
2.1 Materials A (for Subheading 3.1)
2.2 Materials B (for Subheadings 3.2, 3.3, 3.4, and 3.5)
3 Methods
3.1 Fixed Imaging (See Subheading 2.1)
3.2 Live Imaging of Compartment-Specific Dyes (See Subheading 2.2)
3.3 Live Imaging of Endosomal Degradation: Magic Red (MR)
3.4 Live Imaging of Endosomal Degradation: DQ-BSA (DQ)
3.5 Live Imaging of Endosomal Degradation: Bodipy-Pepstatin (BPA)
4 Notes
References
Chapter 37: Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Vesicle Preparation and Immunoprecipitation
2.3 SDS-PAGE and Immunoblotting
2.4 Antibodies
3 Methods
3.1 Cell Seeding
3.2 Cell Lysate Preparation for Immunoprecipitations
3.3 Preparation of Immunobeads for Immunoprecipitations
3.4 Immunoprecipitations
3.5 SDS-PAGE and Transfer onto PVDF Membranes
3.6 Western Blot Decoration with Anti-γ Adaptin Antibodies
3.7 Western Blot Redecorations with Anti-μ1A/B and CHC Antibodies
4 Notes
References
Chapter 38: Live Imaging of Golgi Outposts in Drosophila Dendritic Arbors
1 Introduction
2 Materials
2.1 Drosophila Reagents
2.2 Solutions and Mounting Reagents
2.3 Microscopy
2.4 Software for Image and Data Analysis
3 Methods
3.1 Live Imaging of Golgi Outposts in Neurons in Intact Larvae
3.2 Quantifying Golgi Outposts in the Class IV ddaC Neurons
3.2.1 Quantification of Golgi Outpost Number and Other Properties from Still Images
3.2.2 Quantification of Golgi Outpost Dynamics from Movies
4 Notes
References
Chapter 39: Targeted Protein Unfolding at the Golgi Apparatus
1 Introduction
2 Materials
2.1 Hydrophobic Tagging to Induce Protein Unfolding
2.2 Destabilization of Golgi-Localized DHFR* Fusion Proteins
2.3 Detection of Quality Control (QC) Phenotypes by Imaging
2.4 Monitoring Levels of QC Substrates by Flow Cytometry
2.5 Pulse Labeling of QC Substrates with SNAP-TMR Ligand
2.5.1 SNAP-TMR Pulse Labeling in Live Cells for Fluorescence Microscopy
2.5.2 In-Lysate Labeling with HaloTag-TMR- and SNAP-TMR Ligand Following SDS-PAGE
3 Methods
3.1 Hydrophobic Tagging for Protein Destabilization
3.2 Destabilization of Golgi Localized DHFR* Fusion Proteins
3.3 Detection of Quality Control Phenotypes by Imaging
3.4 Monitoring Levels of QC Substrates by Flow Cytometry
3.5 Pulse Labeling of QC Substrates with SNAP-TMR Ligand
3.5.1 SNAP-TMR Pulse Labeling in Live Cells for Fluorescence Microscopy
3.5.2 In-Lysate Labeling with HaloTag-TMR- and SNAP-TMR Ligand Following SDS-PAGE
4 Notes
References
Part V: Golgi Functions in Physiology and Pathology
Chapter 40: Modeling N-Glycosylation: A Systems Biology Approach for Evaluating Changes in the Steady-State Organization of Go...
1 Introduction
2 Materials
2.1 Preparation of N-glycan Profiles From Whole Cell Lysates
3 Methods
3.1 Preparing Experimental N-glycan Profiles for the SSA Model
3.2 Initializing Enzyme Parameters for Simulating N-glycan Profiles From Altered Cell Line
3.3 Fitting SSA Enzyme Parameters to Simulate Altered Cell Line N-glycan Profiles
3.4 Assessing Predicted Changes in Golgi Organization in Altered Cell Line
4 Notes
References
Chapter 41: Simple N-Glycan Profile Analysis Using Lectin Staining, Mass Spectrometry, and GlycoMaple
1 Introduction
2 Materials
2.1 Glycotransferase Disruption
2.2 Lectin Staining
2.3 Mass Spectrometry
2.4 Predict Glycan Structure by GlycoMaple
2.5 Equipment
3 Methods
3.1 Disruption of Glycan-Related Genes
3.1.1 Transfection
3.1.2 Cell Sorting
3.1.3 Limiting Dilution
3.2 Lectin Staining
3.3 N-glycan Profile Analysis by MALDI-TOF-MS
3.3.1 Release of N-glycans by PNGase F from Whole Cell Lysate
3.3.2 Release of N-glycans From Cell Surface Protein
3.3.3 Release of N-glycans by PNGase F from PVDF Membrane
3.3.4 Clean Up N-glycans with a Sep-Pak C18 Cartridge
3.3.5 Permethylation (See Note 11)
3.4 Glycosylation Mapping Tool (GlycoMaple)
4 Notes
References
Chapter 42: In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Isolation of Cytosol from MDCK II Cells
2.3 Gradients
2.4 Incubation of Golgi Fractions
2.5 Analysis of Incubation Products
2.6 Cytosol Isolated from Pig Brain
2.7 Cell Counting
3 Methods
3.1 MDCK Cell Culture
3.2 Isolation of Enriched Golgi Fractions from MDCK Cells by Ultracentrifugation of PNS in a Sucrose Step Gradient
3.3 Alternative Golgi Fractionation Protocol if Other Organelles Will Also Be Collected
3.4 Further Enrichment of Golgi Vesicles and Dilution of Sucrose
3.5 Isolation of Cytosol from Pig Brain
3.6 Isolation of Cytosol from the MDCK II Cell Line
3.7 Uptake of PAPS into Golgi Vesicles
3.8 Synthesis of GAGs In Vitro by Golgi Fractions in the Presence of Cytosol
4 Notes
References
Chapter 43: Analysis of Golgi Protein Acetylation Using In Vitro Assays and Parallel Reaction Monitoring Mass Spectrometry
1 Introduction
2 Materials
2.1 Purification of Catalytic Domains of Acetyl Transferases and Golgi Proteins
2.2 In Vitro Acetylation Assay
2.3 Detection of Acetylated Proteins by Immunoprecipitation and Western Blotting
2.4 Detection of Acetylated Peptides by Immunoprecipitation and Mass Spectrometry
2.5 Quantification of Acetylated Peptides Using Parallel Reaction Monitoring (PRM)
3 Methods
3.1 Expression and Purification of Acetyl Transferases and GRASP55
3.2 In Vitro Acetylation Assay
3.3 Detection of Acetylated Proteins by Immunoprecipitation and Western Blotting
3.4 Detection of Acetylated Peptides by Immunoprecipitation and Mass Spectrometry
3.4.1 SDS-PAGE and Enzymatic Digestion of Proteins
3.4.2 Guidelines for the Identification of Peptides Using LC-MS/MS
3.5 Quantification of Acetylated Peptides Using Parallel Reaction Monitoring (PRM)
4 Notes
References
Chapter 44: Identification and Characterization of GRASP55 O-GlcNAcylation
1 Introduction
2 Materials
2.1 Materials for Cell Culture and Transfection
2.2 Materials for Immunoprecipitation and Western Blotting
2.3 Materials for Bacterial Transformation and Protein Purification
3 Methods
3.1 Immunoprecipitation and Western Blotting to Detect GRASP55 O-GlcNAcylation
3.2 Determine the O-GlcNAcylation Profile of GRASP55 After Glucose Deprivation
3.3 Preparation of O-GlcNAcylated GRASP55 by Co-expression with OGT in E. coli
4 Notes
References
Chapter 45: Assaying Sterol-Regulated ER-to-Golgi Transport of SREBP Cleavage-Activating Protein Using Immunofluorescence Micr...
1 Introduction
2 Materials
2.1 Cells and Cell Culture Medium
2.2 Cell Plasmid Transfection
2.3 Cell Treatment Medium
2.4 Immunocytochemistry
3 Methods
3.1 SCAP ER-to-Golgi Transport in CHO Cells
3.1.1 Cell Seeding
3.1.2 Cell Treatment
3.1.3 Immunocytochemistry
3.2 SCAP ER-to-Golgi Transport in Transfected HeLa Cells
3.2.1 Cell Seeding for Transfection
3.2.2 Transfection
3.2.3 Cell Seeding for Microscopy
3.2.4 Cell Treatment
3.2.5 Immunocytochemistry
4 Notes
References
Chapter 46: Analysis of Golgi Morphology Using Immunofluorescence and CellProfiler Software
1 Introduction
2 Materials
2.1 Equipment
2.2 Materials
2.3 Reagents
2.4 Recipes
3 Methods
4 Notes
References
Chapter 47: Analysis of Golgi Secretory Functions in Cancer
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cell Culture Reagents
2.3 Live-Cell Assay Reagents
2.4 Perturbation Reagents
2.5 Fixation, Permeabilization, and Blocking Reagents
2.6 Antibodies
2.7 Other Reagents and Kits
2.8 Counterstain, Mounting Media, and Sealants
2.9 Microscopes
2.10 Computer and Software
3 Methods
3.1 Analysis of Golgi Morphology and Function in Cancer Cells Using High-Resolution Fluorescence Microscopy
3.1.1 Golgi Apparatus Morphometry in Fixed Cells
3.1.2 Analysis of Golgi Apparatus Dynamics in Live Cells
3.1.3 Analysis of Golgi-Dependent Secretory Functions
3.2 Analysis of Golgi Secretory Functions in Cancer Cells Using Proteomics and Biochemical Enrichment of Golgi Fractions
3.2.1 Analysis of the Cancer Cell Secretome
3.2.2 Enrichment of Golgi and Vesicle Fractions
4 Notes
References
Index